Semiochemical

ABSTRACT

cis-Jasmone has been discovered to be useful as a semiochemical that changes the behaviour of insects and/or the physiology of plants. It has direct signalling roles with plant-feeding aphids, in attraction of aphid predators and parasitoids, and may act as an airborne signal inducing production of volatile plant semiochemicals, including the monoterpene (E)-β-ocimene, that stimulate foraging by parasitoids. It is an extremely benign compound having, to human beings, a pleasant aroma and gives a long-lasting effect after removal of the stimulus.

[0001] This invention relates to a new use of a material as asemiochemical, for example a plant stress signal, and to a method ofchanging the behaviour of insects and/or the physiology of plantscomprising the exposure of the insects or plants to this material.

[0002] Methyl salicylate has been reported as repelling the black beanaphid, Aphis fabae, and cereal aphids including the grain aphid,Sitobion avenae, and also inhibiting attraction to their host plants.See J. Chem. Ecol. 20, 2565-2574 (1994) (Pettersson et al.) and J. Chem.Ecol. 20, 2847-2855 (1994) (Hardie et al.). It was suggested that thisrepellency arose from the relationship of methyl salicylate withsalicylic acid and inducible plant defence mechanisms, with themetabolite methyl salicylate acting as a volatile and thereby externalsignal; the presence of methyl salicylate signalled that chemicaldefence was induced, and the otherwise attractive host plants were thusperceived as unsuitable hosts by the aphid pests.

[0003] More recently, it has been shown that methyl salicylate also actsas an airborne signal mediating plant pathogen resistance. See Nature385, 718-721 (1997) (Shulaev et al.). Methyl salicylate was initiallyidentified by Pettersson et al. as an aphid semiochemical (for example abehaviour-controlling chemical or a signal otherwise influencing thephysiology of the organism) by gas chromatography (GC) coupled directlyto a single cell recording (SCR) from the olfactory organs on theantenna. Subsequently, more than thirty species of insects, bothplant-feeders and their natural enemies, from four orders have beenfound to possess highly specific and sensitive olfactory receptors forthis compound.

[0004] WO-A-91/19512 (Washington State University Research Foundation)discloses a method of inducing plant defence mechanisms using jasmonicacid or its esters, or substituted derivatives thereof. The compoundinduces the production of plant defence proteins, such as proteinaseinhibitors, and can promote insect, pathogen or viral resistance inplants by inducing the expression of plant defence genes. Plants may becontacted with the compound by direct application to plant tissue or byairborne transmission of the compound. The expression of plant defenceproteins is useful in protecting the plants from the effects of insectattack, but does not prevent the insects in question from attacking theplants. The plants, together with any adjacent plants, will continue tobe attacked by predators. The effect moreover is generally short-livedand disappears after removal of the stimulus.

[0005] Jasmonic acid and methyl jasmonate, along with a, number of othermaterials, are also discussed by Karban and Baldwin in Induced Responsesto Herbivory 1246 (The University of Chicago Press, Chicago, 1997).

[0006] Another material, cis-jasmone, is well known as a volatilecomponent of plants and its release can be induced by damage, forexample during feeding on cotton by lepidopterous larvae. See J. Chem.Ecol., 21, 1217-1227 (1995) (Loughrin et al.). It is a fragrant materialand has often been used for this desirable property. U.S. Pat. No.4,788,164 (Che et al./Hoechst Celanese Corporation) discloses asustained release composition including a fragrance or an insectrepellent. One example (example IV) uses a solution containing jasmoneto impart the odour of jasmine.

[0007] U.S. Pat. No. 5,665,344 (Pair et al./The United States of Americaas represented by the Secretary of Agriculture) indicates thatcompositions of cis-jasmone were found to attract adult Lepidoptera. Thecis-jasmone may be used alone or in combination with one or more othervolatiles of the Japanese honeysuckle flower, particularly linalooland/or phenyl-acetaldehyde. By attracting the adult Lepidoptera toattracticidal baits and/or field traps, the attractants are said to beuseful for the control and monitoring of these agricultural pests. Thecis-jasmone may be combined with an insect toxicant or pesticide to killthese pests.

[0008] We have now discovered that cis-jasmone also has directsignalling roles with plant-feeding aphids, in attraction of aphidpredators and parasitoids, and as an airborne signal inducing productionof volatile plant components, including the monoterpene (E)-β-ocimene,that stimulate foraging by parasitoids. This signalling role isqualitatively different from that of the biosynthetically related methyljasmonate and gives a long-lasting effect after removal of the stimulus.In contrast to what the prior art suggests, it may be used to attractinsects which are beneficial to the plants concerned or to repelundesirable insects.

[0009] Thus according to the present invention there is provided the useof cis-jasmone as a semiochemical that changes—

[0010] the behaviour of insects by acting as an attractant forbeneficial insects and/or repellent of undesirable insects; and/or

[0011] the physiology of plants.

[0012] A semiochemical may be regarded as a chemical signal causing abehavioural change or some other physiological change but withoutgenerally being directly involved in that process.

[0013] The invention may involve the use of cis-jasmone as a plantstress signal or otherwise, and is particularly applicable whencis-jasmone is used on plants so as to cause insect repellency orattractancy. However, cis-jasmone may also be used alone and still causeinsect repellency or attractancy. The terms “beneficial” and“undesirable”, when used in connection with insects, indicate eithertheir desirability to man or, when the cis-jasmone is used on plants,their desirability to the plant concerned. However, when the cis-jasmoneis used on plants to change the physiology of those plants, theinvention is not restricted to the ability of the compound to attractbeneficial insects or repel undesirable insects.

[0014] cis-Jasmone may be used as a repellent of plant-feeding insects,such as plant-feeding aphids. It is however particularly useful as anattractant of beneficial insects, including insect predators and insectparasitoids, especially predators or parasitoids of plant-feedingaphids. It may also, by signifying a plant under stress, encourage someherbivores to attack the apparently weakened plant.

[0015] cis-Jasmone may be used to induce the production of volatileplant semiochemicals, such as (E)-β-ocimene, (E,E)-α-farnesene,(−)-β-caryophyllene and (E)-4,8-dimethyl-1,3,7--nonatriene. We havefound that (E)-p-ocimene is particularly prevalent following exposure ofplants to cis-jasmone, especially when it is used to induce theproduction of volatile semiochemicals from bean plants.

[0016] In a further aspect of the invention, we provide a method ofchanging—

[0017] the behaviour of insects by acting as an attractant forbeneficial insects and/or repellent of undesirable insects; and/or

[0018] the physiology of plants;

[0019] comprising the exposure of the insects or plants to cis-jasmone.

[0020] The cis-jasmone may be applied direct to plant tissue by foliarapplication, but preferably plants are exposed to air containingcis-jasmone. The cis-jasmone is used at a concentration of 10-1000 μgper litre of air, but particularly favourable results are obtained whenthe cis-jasmone is used at a concentration of 50-200 μg per litre ofair, more particularly 75-125 fig per litre of air and especially in theregion of 100 μg per litre of air. This might be achieved by putting thecis-jasmone in an encapsulated form for aerial release.

[0021] The cis-jasmone may lead to the repelling or attraction ofinsects to the plants, for example the repelling of plant-feedinginsects such as aphids. It is particularly useful when it leads to theattraction of insect predators or insect parasitoids, especiallypredators or parasitoids of plant-feeding aphids. This signal would beof immense value for the many areas in which it would be preferable toregulate gene expression, i.e. switch genes on, for the variousrequirements of crop protection, nutrition or yield timing. Attentionmay be drawn to the book, Induced Responses to Herbivory (The Universityof Chicago Press, Chicago, 1997) by Karban and Baldwin, where the needfor plant-defence inducing signals such as that forming the subject ofthe present invention is emphasized. Using a signal will result in plantenergetics not being wasted. Problems with pest resistance would beobviated by useful genes only expressing their products when required.

[0022] Thus, the invention includes the use of cis-jasmone as asemiochemical that changes the behaviour of insects and/or thephysiology of plants, wherein the cis-jasmone is used to effect geneexpression.

[0023] In order that the invention may be better understood, it will nowbe described with reference to the following drawings in which—

[0024]FIG. 1 shows back transformed mean numbers of cereal aphids per 60tillers of winter wheat, Triticum aestivum, following treatment withcis-jasmone on May 5 and Jun. 11, 1999, with an asterisk (*) denotingsignificant difference between treatments;

[0025]FIG. 2 shows levels of (E)-β-ocimene produced by bean plants,Vicia faba (Fabaceae), during 48 hour entrainments following 24 hourexposure to cis-jasmone (100 μg/I in air);

[0026]FIG. 3 shows for comparison levels of (E)-β-ocimene produced by V.faba during 48 hour entrainments following 24 hour exposure to methyljasmonate (100 μg/I in air); and

[0027]FIG. 4. shows differential expression of a gene-specific sequence(D251) in Vicia faba plants. Total RNA was isolated from stem or leaftissue of plants exposed to air, methyl jasmonate or cis-jasmone. 10 μgRNA per sample was loaded and separated on a formaldehyde gel (Panel A).The samples were then transferred to a nylon membrane and probed withthe D251 sequence (Panel B). Lane 1=air treatment (control); lane2=cis-jasmone treatment; lane 3=methyl jasmonate treatment. In the caseof RNA isolated from leaf tissue, only cis-jasmone treatment results inupregulation.

[0028] cis-Jasmone was investigated for behavioural activity with thealate forms of the lettuce aphid Nasonovia ribis-nigri (Homoptera:Aphididae) in a 4-way olfactometer, and was found to be significantlyrepellent (the mean number of entries into the treated arm was 2.0±0.58,whereas the mean number of entries into the control arms was 4.3±0.58;the mean time spent in the treated arm 0.5±0.16 minutes, whereas themean time spent in the control arms was 2.1±0.36 minutes; P=<0.05).Repellency was also demonstrated in preliminary field trials with summermorphs of the hop aphid, Phorodon humuli, where catches in water trapswith visual (yellow) attractancy were reduced by 40% (P<0.04) through aslow release of cis-jasmone (2.05 μg/day/trap).

[0029] Field-trapping experiments were also conducted using cis-jasmoneagainst pollen beetles. Attractive yellow bowl traps, at 1 m height and10 m spacing, were placed in a Latin square design (one row of the Latinsquare=one replicate; traps are re-randomized to the next row of thesquare when a mean of 10 beetles are caught per trap). Catches in anunbaited bowl were compared with those bowls baited with cisiasmonereleased at two different rates. Analysis was by ANOVA, on transformedtotal catch data. The transformation used was x=log₁₀((y+1), where x andy are the transformed and untransformed data, respectively. Transformedmeans were compared using the LSD (least standard difference) test andtransformed back to give the results as set out in Table 1. TABLE 1Field-trapping experiments using cis-jasmone against pollen beetles.Back-transformed mean catch per replicate Experiment A Experiment BAnalysis A unbaited trap 48.3^(a) 333.1^(a) 127.3^(a) cis-jasmone38.2^(ab) 130.7^(bc) 70.9^(b) 2.2 mg/day (21%) (60%) (44%) cis-jasmone31.4^(b) 84.6^(c) 51.7^(c) 25 mg/day (35%) (75%) (59%)

[0030] The means in the same column, followed by different letters, aresignificantly different. P<0.05. Numbers in brackets are the percentreduction in trap catch compared with the unbaited trap.

[0031]FIG. 1 shows the comparison of mean numbers of cereal aphids oncis-jasmone treated and untreated plots in field studies on fivesampling dates. The data have been transformed back from the logs toease presentation. Aphid numbers were consistently lower in thecis-jasmone plots and differed significantly from the control on thelast two sample dates. The predominant aphid species was Metropolophiumdirhodum, the rose-grain aphid. S. avenae and Rhopalosiphum padi werealso present, but numbers were very low. Numbers of parasitized aphidswere also low and no significant difference was observed betweentreatments.

[0032] Since semiochemicals acting as repellents or inhibitors of hostattractancy for herbivorous insects are often involved in predation orparasitism, the activity of cis-jasmone was also investigated at highertrophic levels. Thus, the seven-spot ladybird, Coccinella septempunctata(Coleoptera: Coccinellidae), an important aphid predator, was shown tobe attracted to a source of the compound in the olfactometer (the meannumber of observations in the treated arm was 4.1±1.55, whereas the meannumber of observations in the control arms 2.4±0.69; P=<0.005).Responses of the aphid parasitoid Aphidius ervi (Hymenoptera:Braconidae), which preferentially attacks aphids colonizing plants inthe Fabaceae, were also investigated in a wind tunnel and demonstratedthat cis-jasmone was attractive in a single choice test (Table 2). Theseexperiments indicate a behavioural role for cis-jasmone in influencinginteractions between plants, herbivorous insects and their predators andparasitoids. TABLE 2 Responses of A. ervi in the wind tunnel tosynthetic compounds (10 μg) on filter paper (single choice test).Stimulus No. parasitoids tested % showing oriented flight cis-jasmone 6453.1^(a) (E,E)-α-farnesene 70 60.9^(a) (E)-β-ocimene 61 48.8^(a) hexanecontrol 45 26.7^(b)

[0033] ANOVA analysis F=16.19, P=<0.01. Values followed by a differentletter are significantly different at P=<0.05 (Tukey multiple comparisontest).

[0034] A possible role for the compound as an airborne plant signal wasinvestigated using the broad bean, V. faba (cv The Sutton). Plants werekept for 24 hours in clean air or in air incorporating cis-jasmone at100 μg/l; subsequently, samples of volatiles released by the plants wereobtained by entrainment over 4 periods of 48 hours, i.e. up to 192 hoursafter the end of the treatment. The cis-jasmone itself was undetectableafter 48 hours. However, levels of (E)-β-ocimene released by the plantsexposed to cis-jasmone increased significantly over the 192 hourentrainment period and, in all 4 samples, were 2½-3 times higher thanthose from the control plants (FIG. 2). There was also, from somereplicates, enhancement of (E,E)-α-famesene, (−)-β-caryophyllene and(E)-4,8-dimethyl-1,3,7-nonatriene. These compounds have all beenimplicated in herbivorous insect-induced production and increasedparasitoid foraging. The nonatriene can also be produced innately byplants imitating damage for defence against herbivores and which arethus attractive to parasitoids. (E)-β-Ocimene and (E,E)-α-famesene wereinvestigated with A. ervi in the wind tunnel and both compounds provedto be attractive in the single choice test (Table 2). This activity, andthe elevated levels of these compounds produced by V. faba aftertreatment with cis-jasmone, suggested that there might be increasedforaging and attraction of A. ervi to the treated plants compared withthe controls. Indeed., in the single choice test, V. faba plants taken48 hours after treatment, when cis-jasmone levels were undetectable,were significantly more attractive to A. ervi in the wind tunnel thanuntreated plants (Table 3a). Furthermore, a wind tunnel experiment inwhich A. ervi were offered a choice of treated or untreated plantsdemonstrated that over 3 times as many parasitoids oriented towards thecis-jasmone treated plant compared to the control (Table 3b). TABLE 3Responses of A. ervi in the wind tunnel to V. faba plants 48 hr afterexposure to cis-jasmone (100 μg/l in air). (a) Single choice test No.Plant treated with parasitoids tested % showing oriented flightcis-jasmone 50 44 hexane control 50 20 χ² = 6.62 (contingency test usingPearson on Genstat). P = <0.01. (b) Dual choice test (treated plantversus control plant) % oriented to No. parasitoids tested treated plantcontrol plant 50 32 10 χ² = 5.762 (χ² test). P = <0.05

[0035] We also investigated the activity of methyl jasmonate with V.faba under the same conditions as for cis-jasmone. In this system,exposure to methyl jasmonate did not significantly increase the levelsof (E)-β-ocimene released (FIG. 3). This demonstrates that cis-jasmone,as an airborne signal, has properties different to those of methyljasmonate. cis-Jasmone is closely related to jasmonic acid, being theproduct of further catabolization, i.e. β-oxidation, dehydration anddecarboxylation, although the exact route is not yet reported. Theresults show that, rather than cis-jasmone being considered as merelyanother lipoxygenase-derived volatile and a sink for jasmonic acid, itshould be viewed as a potentially important airborne plant signalrelating to other aspects of plant signalling. It should also be notedthat cis-jasmone is more volatile than methyl jasmonate and, as such,could make a more effective signal compound. We have demonstrated that,far from being biologically inactive, cis-jasmone has activity at allthree trophic levels investigated in this study.

[0036] We have therefore identified a compound capable of inducingproduction of these types of compounds as an airborne signal, namelycis-jasmone, an extremely benign compound having, to human beings, apleasant aroma.

Methods

[0037] Electrophysiology.

[0038] Electroantennogram (EAG) recordings from alate N. ribis-nigriwere made using Ag—AgCl glass electrodes filled with saline solution, asreported in J. Exp. Biol. 51, 71-97 (1969) (Maddrell) but without theglucose. The insect was anaesthetized by chilling and the head wasexcised and mounted on the indifferent electrode. The tip of therecording electrode was removed so that its inside diameter was justwide enough to accept the terminal process of the antenna. The signalswere passed through a high impedance amplifier (UN-03b, Syntech) anddisplayed on an oscilloscope.

[0039] Coupled Gas Chromatography (GC)-Electrophysiology.

[0040] The coupled GC-electrophysiology system, in which the effluentfrom the capillary column GC is delivered simultaneously to the antennalpreparation and the GC detector, has been described previously. SeeWadhams in Chromatography and Isolation of Insect Hormones andPheromones (eds. McCaffery el al.) 289-298 (Plenum Press, New York,1990). Separation of the air entrainment sample was achieved on an Al 93GC equipped with a cold on-column injector and a flame ionizationdetector (FID). The column (30 m×0.53 mm ID, HP-1) was maintained at 40°C. for 2 min and then programmed at 10° C./min to 250° C. The carriergas was hydrogen. The outputs from the EAG amplifier and the FID weremonitored simultaneously on a chart recorder.

[0041] Olfactometry: aphids.

[0042] Behavioural assays were done in a Perspex™ olfactometer similarto that described in J. Entomol. Scand 1, 63-73 (1970) (Pettersson),with a weak airstream directed towards the centre from each of 4 sidearms. The test compound (1 μg) in hexane (10 μl) was placed on filterpaper (Whatman No. 1) at the end of one of the side arms, with hexanealone used as a control in the other arms. One alate virginopara of N.ribis-nigri was placed in the centre of the arena and its movementsobserved over 10 min. The apparatus, maintained at 24° C., was lit fromabove by fluorescent tubing and was rotated 90° every 2.5 min to avoidany directional bias. The experiment was replicated 6 times and resultsanalysed by Student's t-test.

[0043] Olfactometry: ladybirds.

[0044] Apparatus and methodology were similar to that employed foraphids (above). The test compound was applied in 0.5 μl microcaps(Drummond Sci. Co.) at the end of one of the side arms and each arm wassupplied with moist filter paper to minimize differences in relativehumidity. Individual C. septempunctala were introduced into the centreof the arena and their positions noted every 2 min for 20 min. Theexperiment was replicated 8 times and results analysed as above.

[0045] Wind Tunnel Studies.

[0046] Naive female A. ervi were flown in a wind tunnel, as described inJ. Chem. Ecol. 16, 381-396 (1996) (Poppy et al.). The parasitoids werereleased 25 cm downwind (single choice tests) or 40 cm downwind (dualchoice test) of the target, which was either a plant or a syntheticcompound (10 μg in 10 μl hexane) placed on a 2×1 cm strip of filterpaper (Whatman No. 1) surrounded by a ring of green crepe paper. Theproportions of parasitoids responding with an oriented flight to thesynthetic chemicals were calculated each day on 3 separate days. Thesevalues were then subjected to a logit transformation to normalize thedata before being analysed by ANOVA followed by Tukey post-hoc tests.The number of parasitoids orienting upwind to the single plant targetwere recorded and subjected to a χ² contingency test (Pearson method onGenstat—see Genstat 5 Committee. Genstat 5 Reference Manual, Release 3(Clarendon Press, Oxford, 1993)) to determine whether an orientingresponse was linked to the type of plant treatment. The numbersorienting to each plant in the dual choice test were analysed by a χ² todetermine whether one plant was more attractive than the other.

[0047] Field Studies.

[0048] Plots (6 m×6 m) of winter wheat, Triticum aestivum (cv Consort),were arranged in a 5×5 quasi-complete Latin square design. The fivecis-jasmone treated plots were sprayed on May 5 and Jun. 11, 1999, usinga hand-held hydraulic device, at a rate of 50 g active ingredient/ha in200 l/ha of aqueous Ethylan BV (0.1%). Control plots were untreated.Cereal aphids and parasitized aphids were counted on 8 occasions betweenearly May and mid-July. At each count, five tillers were inspected at 12separate sites on two diagonal transects totalling 60 tillers per plot.Transformed data (y=log(y+1)) were subjected to ANOVA and the sums ofsquares of the treatments were partitioned to test for significantdifferences.

[0049] Induction Studies.

[0050] Bean plants, V. faba (cv The Sutton) were grown under standardglasshouse conditions until the 24 leaf stage when they were rinsed freeof soil and transplanted into baked glass jars containing washed sand,with 3 plants per jar. These were left for 1-2 days to acclimatize.Plants to be treated (three jars of three plants each) were sealed in a25 l glass tank for 24 hours with either cis-jasmone or methyl jasmonate(2.5 mg) applied to a piece of filter paper (Whatman No. 1) placed onthe floor of the tank. Treated or untreated plants were then placed in10 l glass entrainment vessels and the volatiles from each collectedover 48 hour periods for 192 hours. See Blight in Chromalography andIsolation of Insect Hormones and Pheromones (eds. McCaffery et al.)289-298 (Plenum, New York, 1990). Volatiles were eluted from glass tubescontaining Porapak Q (50 mg), using freshly distilled diethyl ether (500μl), and then concentrated to 100 μl for analysis by GC and GC-MS.

[0051] Analysis.

[0052] GC analysis was carried out using a Hewlett Packard 5890 GCequipped with a temperature programmable on-column injector and FID.This was fitted with HP-1 (50 m×0.32 mm ID) and SPB-35 (30 m×0.32 mm ID)columns with hydrogen as the carrier gas. The oven was maintained at 40°C. for 1 min then programmed at 10° C./min to 250° C. GC-MS analysis wascarried out using a Hewlett Packard 5890 GC connected to a VG Autospecmass spectrometer (Fisons Instruments). Ionization was by electronimpact at 70 eV, 230° C. The GC was maintained at 30° C. for 5 min thenprogrammed at 5° C./min to 180° C. Detection limits for cis-jasmone inthe entrainment samples were 40 pg/hour for GC and 400 pg/hour forGC-MS. Compounds identified by GC/GC-MS were confirmed by co-injectionof authentic samples on HP-1 (non-polar) and SPB-35 (polar) columns.Authentic samples were obtained from commercial sources, except for(E)-β-ocimene, (E,E)-α-farnesene and (E)-4,8-dimethyl-1,3,7-nonatrienewhich were synthesized by standard methods, as follows.

[0053] (E,E)-α-Farnesene and (E)-β-ocimene.

[0054] (E,E)-α-Farnesene and (E)-β-ocimene were synthesized from3-methyl-2,5--dihydrothiophene-1,1-dioxide. See J. Chem Soc. Chem.Comm., 1984, 1323 (Chou et al.). Sulphur dioxide elimination wasachieved using excess lithium aluminium hydride following a modifiedprotocol based on that in Tetrahedron Lett., 1977, 11, 947 (Gaoni).

[0055] To a stirred suspension of lithium aluminium hydride (1 equiv. byweight) in refluxing dry diethyl ether (10 mmol/ml) was added dropwisevia syringe a solution of the dihydrothiophene-1,1-dioxide (1 equiv.) indry diethyl ether (1 ml). After refluxing for 1 hour, the cooled (0° C.)mixture was treated with 15% NaOH (1 ml), water (3 ml), and the mixturefiltered through Celite™. Evaporation of the filtrate under reducedpressure followed by column chromatography over Florisil (100% hexane)yielded the product as a colourless oil.

[0056] 4,8-Dimethyl-1,3,7-nonatriene.

[0057] 4,8-Dimethyl-1,3,7-nonatriene was synthesized in two steps fromgeraniol.

[0058] Geraniol (7.0 g, 0.045 mol, 1 equiv.), manganese (IV) dioxide(100.0 g) and dichloromethane (500 ml) were stirred together at ambienttemperature overnight. The mixture was filtered through Celite™, and thefiltrate concentrated in vacuo to yield geranial (5.42 g, 80%). A cooled(−15° C.), stirred suspension of methyltriphenyl-phosphonium iodide(16.0 g, 0.039 mol, 1.1 equiv.) in tetrahydrofuran (50 ml) and diethylether (50 ml) was treated with n-butyllithium (2.5 M; 16 ml, 0.039 mol,1.1 equiv.). After 0.25 hours, geranial (5.42 g, 0.036 mol, 1 equiv.)was added and the mixture allowed to stir at ambient temperature for 1hour. The mixture was partitioned between diethyl ether (200 ml), water(200 ml) and petroleum ether b.p. 40-60° C. (200 ml), and the organiclayer dried (MgSO₄) and concentrated in vacuo to yield a crude oil whichwas subjected to column chromatography over Florisil (100% hexane) togive the nonatriene as a colourless oil (3.42 g, 64%).

Differential Display

[0059] In order to determine if cis-jasmone was capable of inducingalterations in plant gene expression, the sensitive technique ofdifferential display was carried out on RNA extracted from plants whichhad been exposed to air, methyl jasmonate or cis-jasmone. A number ofthe resulting PCR products were observed to show alterations in theirabundance in the presence of cis-jasmone. To confirm this observation,bands of interest were recovered by excision from the dried gel andre-amplified with the appropriate pair of oligonucleotide primers. Theresulting PCR products were cloned and sequenced to confirm thehomogeneous nature of the amplified product. These sequences were thenused to probe RNA isolated from control or treated V. faba plants. Ascan be seen from FIG. 4, one particular sequence (D251) was shown to beupregulated in leaf tissue only in the presence of cis-jasmone.Interestingly, when this same (cloned) sequence was used to probe RNAisolated from V. faba stem tissues, it was upregulated to a similarlevel in plants that had been treated with either cis-jasmone or methyljasmonate. It is important to note that the nature of the differentialdisplay technique generates short gene-specific probes, containingmainly 3′ untranslated regions of transcribed sequences, and thereforethe functional part of the differentially expressed gene is unknown.

[0060] Thus, using differential display and confirmatory northernblotting, we have shown that methyl jasmonate and cis-jasmone haveapparently distinct effects on plant gene expression. In this study, thedifferentially displayed PCR product D251 was cloned and used to probenorthern blots from leaf or stem tissues of V. faba plants treated withair, methyl jasmonate or cis-jasmone. This clearly showed (FIG. 4) thatwhilst the D251 sequence was upregulated by treatment with vapours ofboth compounds in stem tissue, only cis-jasmone brought about anincrease in the steady-state transcript level of this sequence in leaftissue. Thus, the two compounds have distinct effect on plant geneexpression and the response to these signalling compounds may betissue-specific.

1. Use of cis-jasmone as a semiochemical that changes— the behaviour ofinsects by acting as an attractant for beneficial insects and/orrepellent of undesirable insects; and/or the physiology of plants. 2.Use as claimed in claim 1, wherein cis-jasmone is used as a plant stresssignal.
 3. Use as claimed in claim 1 or 2, wherein cis-jasmone is usedalone or on plants so as to cause insect repellency or attractancy. 4.Use as claimed in claim 3, wherein cis-jasmone is used as a repellent ofplant-feeding insects.
 5. Use as claimed in claim 4, wherein the insectsare plant-feeding aphids.
 6. Use as claimed in claim 3, whereincis-jasmone is used on plants as an attractant of beneficial insects. 7.Use as claimed in claim 6, wherein cis-jasmone is used as an attractantof insect predators or insect parasitoids.
 8. Use as claimed in claim 7,wherein the insect predators or insect parasitoids are predators orparasitoids of plant-feeding aphids.
 9. Use as claimed in any precedingclaim, wherein cis-jasmone is used to induce the production of volatileplant semiochemicals.
 10. Use as claimed in claim 9, wherein thevolatile plant semiochemicals comprise one or more compounds selectedfrom (E)-β-ocimene, (E,E)-α-farnesene, (−)-β-caryophyllene and(E)-4,8-dimethyl-1,3,7-nonatriene.
 11. Use as claimed in claim 10,wherein the volatile plant semiochemicals comprises (E)-β-ocimene. 12.Use as claimed in any one of claims 9 to 11, wherein the cis-jasmone isused to induce the production of volatile semiochemicals from beanplants.
 13. A method of changing— the behaviour of insects by acting asan attractant for beneficial insects and/or repellent of undesirableinsects; and/or the physiology of plants; comprising the exposure of theinsects or plants to cis-jasmone.
 14. A method as claimed in claim 13 inwhich the insects or plants are exposed to air containing cis-jasmone.15. A method as claimed in claim 13 in which the cis-jasmone is used ata concentration of 10-1000 μg per litre of air.
 16. A method as claimedin claim 15 in which the cis-jasmone is used at a concentration in theregion of 100 μg per litre of air.
 17. A method as claimed in any one ofclaims 13 to 16, wherein cis-jasmone is used alone or on plants to leadto the repelling or attraction of insects.
 18. A method as claimed inclaim 17, wherein cis-jasmone is used to lead to the repelling ofplant-feeding insects.
 19. A method as claimed in claim 18, wherein theinsects are plant-feeding aphids.
 20. A method as claimed in claim 17,wherein cis-jasmone is used on plants to lead to the attraction ofinsect predators or insect parasitoids.
 21. A method as claimed in claim20, wherein the insect predators or insect parasitoids are predators orparasitoids of plant-feeding aphids.
 22. A method as claimed in any oneof claims 13 to 21, wherein the production of volatile plantsemiochemicals is induced.
 23. A method as claimed in claim 22, whereinthe volatile plant semiochemicals comprise one or more compoundsselected from (E)-β-ocimene, (E,E)-α-farnesene, (−)-β-caryophyllene and(E)4,8-dimethyl-1,3,7-nonatriene.
 24. A method as claimed in claim 23,wherein the volatile plant semiochemicals comprises (E)-β-ocimene.
 25. Amethod as claimed in any one of claims 22 to 24, wherein bean plants areexposed to cis-jasmone.
 26. Use as claimed in any one of claims 1 to 12,wherein the cis-jasmone is used to effect gene expression.